Alterations in DNA repair play major roles in breast cancer resistance to ionizing radiation (IR) and chemotherapies. Factors operating in both DSB repair and RNA transcription, specifically proteins regulating RNA polymerase (Pol) II, have been suggested, but few reported. When RNA Pol II stalls, or when transcription cannot properly terminate, RNA/DNA hybrids (R-loops) have extended half-lives that result in DNA double strand break (DSB) formation and genetic instability. Using yeast two hybrid analyses, we identified Ku70 binding protein #5 [(Kub5)/Hera], a highly conserved human homolog of yeast rtt103 that terminates RNA transcription via its regulation of RNA Pol II. Stable loss of Kub5/Hera expression, either by heterozygote knockout or knockdown by siRNA/shRNA expression, leads to elevated basal DSBs, ATM activation, foci formation and chromatid aberrations that were abrogated by RNase H forced over- expression, suggesting a role for R-loops in genetic instability. Kub5/Hera knockdown cells were hypersensitive to IR, equivalent to Ku70 deficient cells, and failed to repair DSBs that require DNA end-processing. We hypothesize that KUB5/HERA plays dual roles in the cell to: (i) regulate RNA Pol II to terminate RNA transcription via its interaction with the RNA Pol II CTD domain; and (ii) stimulate NHEJ processing of complex DSBs by Ku70 and Artemis interactions. When its expression is depressed (e.g., one-half via haplo-insufficiency) R-loops form and DSBs are created due to deficient RNA transcription termination. DNA damage is simultaneously amplified by the cell's inability to repair DSBs downstream, leading to IR hypersensitivity. Three Specific Aims are proposed: Aim 1: to perform structure/function analyses of Kub5/Hera focusing on two regions, the RPR and coiled-coil domains, to uncouple transcription termination and DSB repair; Aim 2: to define functions of KUB5- Ku70-Artemis complexes in DSB repair; and Aim 3: to define the role(s) of KUB5/HERA in regulating RNA Pol II function, RNA transcription termination, and spontaneous DSB formation and chromatid aberrations. These studies will allow us to explore the role of KUB5/HERA over-expression in human breast cancer radio-resistance.